The invention generally relates generally to the fields of biology, chemistry, and biochemistry and, more particularly, to devices and methods for performing multiple biological, biochemical, and chemical assays in small volumes.
In the fields of biology, chemistry, and biochemistry, often subtle changes to a molecule or a cell in a reaction can have consequences on the results of reactions or assays using that molecule or cell. For example, changing a single amino acid (e.g., an alanine to a serine) in a polypeptide molecule or changing the growth conditions of a cell (e.g., growth in the presence of 10% fetal bovine serum versus 5% fetal bovine serum) can affect how the polypeptide or cell responds in a given reaction (e.g., whether or not the polypeptide binds to a specific binding agent or whether the cell responds to a growth factor).
Microtiter plates have been used for decades to enable the multiple reactions in small volume for applications including high-throughput screening. For example, one commonly used immunological assay, the Enzyme-linked immunosorbent assay or “ELISA” can be used to determine if a member of a binding agent/ligand pair is present in a sample, and, if present, how much is present. For example, one member of the binding agent/ligand pair (e.g., an antibody) can be immobilized to the bottom of the multiple wells on a microtiter plate (e.g., through non-specific adsorption of the antibody into the wells of the plate), and then multiple samples can be assessed as to the presence and/or concentration of the antibody's specific ligand by adding each different sample to one of the wells of the microtiter plate and then detecting binding of the sample to the well (e.g., using a detectably labeled antibody specific for the ligand).
Standard microtiter plates are commercially available from numerous manufacturers (e.g., Thermo Fisher Scientific, Waltham, Mass.) and can be made from numerous different materials (see, e.g., Bouche, F B et al., Clinical Chemistry 48: 378, 380, 2002). However, standard microtiter plates have several limitations. Most relevantly, they are limited by the number of wells on the plate. To increase the number of reactions that can be run (e.g., increase the number of samples that can be tested at the same time), the number of wells on a single plate can vary from 6 wells to 1536 wells per plate.
Recently, in the field of biology and biochemistry, DNA microarrays and protein microarrays have been employed to increase still further the number of different reactions that can be performed simultaneously. DNA microarrays are made by adhering DNA probes (e.g., single-stranded probes) to the surface of a chip or slide (e.g., made of glass or silicon) in an array of dots or spots. Different samples of DNA are then added to each of the spots and screened for the ability to bind the spots (e.g., through hybridization of a nucleic acid in the sample to the surface-bound probe). Detection of binding can then be made, for example, by fluorescent or chemical means (which, in some cases, is preceded by amplification of the bound nucleic acid molecules to enhance detection). DNA microarray technology is well known (see, e.g., U.S. Pat. Nos. 5,700,637; 7,323,555; 6,862,363; 7,414,117; and 7,359,537).
Of course there are some fundamental differences between microarrays and microtiter plates. In microtiter plates, a reaction in an individual well can be carried out independently regardless of a reaction in the neighboring wells. In contrast, the active spots (similar to ‘wells’ of a microtiter plate) in microarrays are usually exposed to a common solution. Unlike micrrotiter plates, microarrays do not offer any capability where an individual spot can be exposed to a different solution during a repeated process of addition, incubation, and washing.
Protein arrays on glass slides have also been described (see Arenkov et al., Anal. Biochem 278: 123-131, 2000; Guschin et al., Anal. Biochem. 250: 202-211, 1997; MacBeath and Schreiber, Science 289: 1760-1763, 2000) as well as protein arrays on microwell or nanowell chips (see Zhu and Snyder, Curr. Opin. Chem. Biol. 5(1):40-45, 2001). However, in addition to having the same limitations as DNA microarrays, protein arrays have additional challenged. For example, complex chemicals, such as proteins and other non-nucleic acid biological molecules (e.g., fatty acids and carbohydrates), are more difficult to use in microarrays for multiple reactions. This is due a variety of actors including, for example, the storage and binding requirements of the molecules (e.g., storage may be preferable at −20° C. while binding may be preferable at 37° C. For these reasons, protein microarrays are generally less specific than assays such as ELISAs that use microtiter plates.
Accordingly, there is a need find a solution for running multiple reactions that can combine the specificity of microtiter plate assays with the microarray's increased number of reactions that can be in multiple.